DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Substrate Design
2.3. Purification of Recombinant Proteins
2.4. Construction, Expression, and Purification of the Catalytic Domain of h-p300
2.5. Acetylation of Recombinant h-Pol δ
2.6. Pol δ Extension, Strand Displacement, and Structure Resolution Assays
2.7. Exonuclease Assays
2.8. Quantification of Polymerization and Exonuclease Products
2.9. Mammalian Cell Culture
2.10. IP and Western Blot Analysis of h-pol δ
2.11. Analysis of the Acetylation Status of h-pol δ by Mass Spectroscopy
3. Results
3.1. DNA Polymerase δ Is Acetylated In Vitro and In Vivo
3.2. Acetylation Stimulates the Synthesis Activity of DNA Polymerase δ
3.3. Acetylation Stimulates the Strand Displacement Activity of Pol δ
3.4. Acetylation Enhances the Ability of Pol δ to Resolve Complex Secondary Structures
3.5. Replication Accessory Proteins Can Stimulate Acetylated Polymerase
3.6. Acetylated Polymerase Displays Higher Exonuclease Activity
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Length | Sequence |
---|---|---|
Upstream (5′-3′) | ||
U1 | 44 | GTCCACCCGACGCCACCTCCTGCCTTCAATGTGCTGGGATCCTA |
U2 | 25 | GTCCACCCGACGCCACCTCCTGCCT |
U3 | 25 | GTCCACCCGACGCCACCTCCTGACT |
U4 | 25 | GTCCACCCGACGCCACCTCCCGCCT |
U5 | 25 | GTCCACCCGACGCCACCTACTGCCT |
Downstream (5′-3′) | ||
D1 | 60 | AGACGAATTCCGGATACGACGGCCAGTGCCGACCGTGCCAGCCTAAATTTCAATCCACCC |
D2 | 60 | GGGCGACTCCCGGGGGCGCCGGCCCGTGCCGGCCGTGCCGGCCTCCCTGTCAACCCACCC |
D3 | 60 | AAACTAATTCTGAATAAGAAAAAAAGTGTTTAACTTAAAAGCCTAAATTTAAATCCAAAA |
Template (3′-5′) | ||
T1 | 110 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTTCTGCTTAAGGCCTATGCTGCCGGTCACGGCTGGCACGGTCGGATTTAAAGTTAGGTGGG |
T2 | 110 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTCCCGCTGAGGGCCCCCGCGGCCGGGCACGGCCGGCACGGCCGGAGGGACAGTTGGGTGGG |
T3 | 110 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTTTTGATTAAGACTTATTCTTTTTTTCACAAATTGAATTTTCGGATTTAAATTTAGGTTTT |
T4 | 80 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTGGTTCTGCCGGACGAGTATTATCTCGTCCGGTAAAGT |
T5 | 110 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTTAGGGTTAGGGTTAGGGTTAGGGGTCACGGCTGGCACGGTCGGATTTAAAGTTAGGTGGG |
T6 | 110 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTTCTGTCCAGAGCTCCGGACGAGTATTATCTCGTCCGGAGCTCTGGATAAAGTTAGGTGGG |
T7 | 80 | CAGGTGGGCTGCGGTGGAGGACGGAAGTTACACGACCCTAGGATGTTGGTTCTGCTTAAGGCCTATGCTGCCGGTCACGG |
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Njeri, C.; Pepenella, S.; Battapadi, T.; Bambara, R.A.; Balakrishnan, L. DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation. Genes 2023, 14, 774. https://doi.org/10.3390/genes14040774
Njeri C, Pepenella S, Battapadi T, Bambara RA, Balakrishnan L. DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation. Genes. 2023; 14(4):774. https://doi.org/10.3390/genes14040774
Chicago/Turabian StyleNjeri, Catherine, Sharon Pepenella, Tripthi Battapadi, Robert A. Bambara, and Lata Balakrishnan. 2023. "DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation" Genes 14, no. 4: 774. https://doi.org/10.3390/genes14040774
APA StyleNjeri, C., Pepenella, S., Battapadi, T., Bambara, R. A., & Balakrishnan, L. (2023). DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation. Genes, 14(4), 774. https://doi.org/10.3390/genes14040774